Identification and quantification of plasma free salusin-β, an endogenous parasympathomimetic peptide

Abstract

Salusin-β is an endogenous parasympathomimetic proatherosclerotic peptide. Salusin-β was initially predicted from bioinformatic analyses and later immunologically detected in human biofluids. However, elucidation of salusin-β bioactivity has faced enormous challenges because of its unique physicochemical characteristics that cause it to strongly adhere to laboratory apparatus materials. In the strictest sense, the discovery of bioactive peptides is not complete until their exact native sequences have been confirmed in the peripheral circulation. In this study, we determined the plasma molecular form and levels of free salusin-β to determine its pathophysiological significance. Ultra-high-yield enrichment and preseparation of non-tryptic human plasma was followed by LC-MS/MS, and full-length salusin-β and seven different endogenous fragment sequences were identified. We established a new ELISA that specifically detects plasma free salusin-β without cross-reacting with any of its identified endogenous fragments. Free salusin-β levels exhibited a profound early morning nadir and rapidly decreased in response to parasympathetic nervous augmentation. Our technical advance in plasma native peptide analysis successfully identified a hard-to-detect bioactive peptide, salusin-β, together with its formerly unrecognized fragments, and further suggests that conventional immunological measurements of target peptides may not be fully representative.

Identification of the salusin-β receptor using proteoliposomes embedded with endogenous membrane proteins

“…Salusin-β has a unique physicochemical property that has discouraged many attempts to unravel its biological activity 24 . Purified synthetic salusin-β shows marked adhesiveness to polypropylene, polystyrene and glass and, when reconstituted and diluted in distilled water in such tubes, may rapidly disappear from the solution before being applied to in vitro or in vivo experiments 14,25,26 . This nature appears to derive partly from the presence of hydrophobic residues at its N-terminal end (Supplementary information), but the problem can be alleviated by the addition of 0.01–0.1% NP40 or Tween 20, thus allowing a fair assessment of its bioactivity 14,26,27 .…”

“…A number of membrane proteins have previously been embedded in artificial liposomes; however, identifying an unknown receptor using endogenous membrane protein-embedded liposomes still remains extremely challenging. Proteoliposome techniques using detergents to extract membrane proteins increases the difficulty of in vitro approaches 11,12 , with detergents affecting the ligand binding capabilities of reconstituted proteoliposomes 13,14 . Moreover, conventional proteoliposomes can aggregate with one another and are difficult to pass through affinity columns.…”

Identification of plasma binding proteins for glucose-dependent insulinotropic polypeptide

“…Synthetic peptides, [Cys 0 ]-PEG-HKHNITQ, YAEGTF-PEG-[Cys 0 ] and EGTFIS-PEG-[Cys 0 ] were pretreated with a protein crosslinking and fixation reagent, mixed with respective untreated peptide, coupled to maleimideactivated mariculture keyhole limpet hemocyanin (Pierce), and immunized into Japanese white rabbits in the laboratory of Scrum Inc. (Tokyo, Japan) as described previously [8]. Polyclonal GIP antiserum and normal human IgG were purified using a Melon TM Gel IgG Purification Kit (Thermo Fisher Scientific, MA, USA) in accordance with the manufacturer's instructions.…”

A novel strategy to identify autoantigens by proteomic analysis of plasma IgG-bound proteins

“…In our original protocol, plasma LMWPs are first denatured, which allows LMWPs bound to carrier proteins to dissociate efficiently. This methodology has enabled detection of low-abundance authentic bioactive peptides that could never have been obtained used conventional methods 9) .…”